Plasma-like solution

ABSTRACT

Aqueous solutions comprising a polysaccharide oncotic agent, a physiologically compatible buffer, a simple hexose sugar, dissolved chloride salts of calcium, sodium and magnesium, and a dissolved organic salt of sodium are disclosed. The solutions are effective substitutes for blood and may be used to preserve the biological integrity of the organs of a mammalian donor organism as shown by superior anatomical integrity of cryopreserved organs and tissues of subjects perfused with the solution. The solutions may be used for maintaining a partially or substantially completely exsanguinated subject at normal temperatures and at temperatures substantially below those normally maintained by a mammal and may be used in conjunction with hypobaric environments to maintain such partially or completed exsanguinated subjects alive without infusing blood back into the subject.

CROSS-REFERENCES

[0001] This patent application is a continuation-in-part of applicationSer. No. 08/133,527 filed Oct. 7, 1993, which is a continuation-in-partof application Ser. No. 08/071,533, filed Jun. 4, 1993, whichapplications are incorporated herein by reference and to whichapplications we claim priority under 35 U.S.C. § 120.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of aqueoussolutions such as plasma-like solutions used to perfuse a living subjectin need of perfusion and which act as effective substitutes for blood.The invention also relates to methods of preserving the biologicalintegrity of the organs of a mammalian donor organism (as shown bysuperior anatomical integrity of cryopreserved organs and tissues ofsubjects perfused with the solution of the invention) and to methods ofmaintaining a partially or substantially completely exsanguinatedsubject at temperatures substantially below those normally maintained bya mammal.

BACKGROUND OF THE INVENTION

[0003] Two clinically applied preservation methods for organs are known:(1) initial perfusion for about 5 min with subsequent cold storage (2°C.), and (2) continuous perfusion using solutions containing albumin orplasma.

[0004] Many of the solutions used for initial perfusion with subsequentcold storage are based on the solutions of Collins et al. (1969) Lancet2:1219 and Sacks et.a. (1973) Lancet 1:1024. Ross et al. (1976)Transplantation 21:498 compared canine renal preservation followingflushing and storage for 72 hours in various solutions. It was foundthat only kidneys preserved in a hypertonic citrate (HC) solution(comprising in part 80 mM K⁺, 55 mM citrate, 400 mosmol/kg, pH 7.1)survived after 72 hours. The Collins and Sacks solutions in partcontained 115-126 mM K⁺, 290-430 mosmol/kg, pH 7.0-7.3. Wall et al.(1977) Transplantation 23:210 reports the hypothermic preservation ofhuman livers for up to about 4 hours in a solution in part comprising250 mg dextrose, and 15 mEq potassium phosphate. Bishop & Ross (1978)Transplantation 25:235 reported that renal function was preserved bestin the HC solution of Ross et al. (1976) supra, rather than otheravailable solutions. Fischer et al. (1985) Transplantation 39:122 founda new preservation solution for hypothermic ischemic storage (comprisingin part 110 mM Na⁺, 115 mM K⁺, 400 mOsm/kg, solvent D2O, 110 mM HEPES)to be superior to other solutions in clinical use, including Collins,Sacks, and HC.

[0005] Among the solutions used for continuous organ perfusion, Belzeret al. (1985) Transplantation 39:118 reported a newly developed solutionwhich preserved renal function when kidneys were perfused for 48 hoursand stored for 24 hours (comprising in part 80 mM sodium gluconate, 22mEq/1 K⁺, 128 mEq/l Na⁺, 4.9 mM adenosine, 10 mM HEPES, 3.0 mMglutathione, 3.75 g% albumin, pH 7.45). Kallerhoff et al. (1985)Transplantation 39:485 examined the effect of temperature on pH oforgans continuously perfused with two different solutions (Euro-Collins:10 mM Na⁺, 115 mM K⁺, 198 mM glucose, 406 mOsm/L, pH 7.2 at 20° C.; HTK:15 mM Na⁺, 10 mM K⁺, 2.0 mM tryptophan, 180 mM histidine, 30 mMmannitol, 310 mOsm/L, pH 7.3 at 8° C.). At incubation temperaturesbetween 5° C.-35° C., HTK solution maintained pH at consistently highervalues than Euro-Collins solution.

[0006] Klebanoff & Phillips (1969) Cryobiology 6:121 describehypothermic asanguinous perfusion of dogs perfused with bufferedRinger's lactate at 7.1 to 16° C. Segall et al. (U.S. Pat. No.4,923,442) describe a blood substitute capable of maintaining a subjectand its organs at temperatures below 20° C. having four differentsolutions—a base solution, a cardioplegia-inducing solution, acardioplegia-maintaining solution, and a recovery solution. The basesolution contains electrolytes in physiological concentration, amacromolecular oncotic agent, a conventional biological buffer effectiveat physiological pH, sugar, and K⁺ ranging from 4-5 mEq. Thecardioplegia-inducing solution had a K⁺ concentration of 25-45 mEq; thecardioplegia-maintenance solution had a K⁺ concentration of 15-45 mEq;and the recovery solution had a K⁺ concentration of 6-10 mEq. Segall etal. (U.S. Pat. No. 5,130,230) further described the four-solutionsystem, where the recovery solution contains 0-10 mEq K⁺.

SUMMARY OF THE INVENTION

[0007] This invention features methods of using a single solutionsuitable to maintain a partially or substantially completelyexsanguinated subject alive at normal temperatures or at temperaturessubstantially below those normally maintained by a mammal, generallyless than 37-38° C. and greater than -20C, comprising a sub- and/orphysiological levels of K⁺ and Mg⁺; physiological Na⁺, Ca⁺⁺, Cl⁻; amacromolecular oncotic agent; an organic carboxylic acid or saltthereof; and a sugar.

[0008] The solution of the invention may be used as a plasma extender atnormal body temperature. The solution of the invention is also useful tomaintain the life or the biological integrity of a perfused subjectand/or its organs during and after exposure to profound hypothermicconditions. The solution can also be used to maintain a euthermicsubject in a pressurized environment with increased oxygen concentrationup to 100% O₂ for time periods sufficient to permit adequate restorationof the subject's blood components.

[0009] The solution according to the invention may be used to perfuseand chill a mammalian subject to temperatures profoundly hypothermic tothe subject's normal temperature. The solution can be used to maintainthe subject in profound hypothermia for long periods of time, usuallyexceeding an hour, from which an intact subject can recover withoutapparent durable ill effects.

[0010] An important distinction of the solution of the present inventionis that it does not require multiple solutions for it to be effectivelyadministered to a subject for the purposes of blood substitution, or lowtemperature maintenance of a mammalian subject. The solution of theinvention may be used at all phases of plasma extension or bloodsubstitution.

[0011] Another important distinction of the solution of the presentinvention is the feature of a subphysiological amount of K⁺ at all stepsof administration. This requirement reduces the risk ofhyperkalemia-induced heart sufficiency resulting in blood transfusion inprimates and humans.

[0012] Another important distinction of the solution of the presentinvention is the absence of a conventional biological buffer. Theabsence of a conventional biological buffer in the solution confers theimportant medical advantage of allowing the solution to be terminallyheat sterilized without degradation of solution components.

[0013] The solution of the present invention requires the presence of anorganic carboxylic acid, salt, or short chain esters thereof. Theorganic carboxylic acid, salt or ester thereof is a component of thedynamic buffer system of the solution able to maintain a biologicallyappropriate pH range when used in a mammal.

[0014] The solution of the present invention requires the presence of amacromolecular oncotic agent sufficient to maintain physiologicalosmotic pressure. The macromolecular oncotic agent used in the solutionof the present invention may be a protein(s) or starch(es).

[0015] An advantage of the solution is that it can be used in amammalian subject during all phases of blood substitution from initialwashout of the subject's blood through full substitution of all orsubstantially all circulating blood.

[0016] A feature of the invention is that it may be used to maintain amammal without blood and also during re-perfusion with blood.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0017] It must be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a formulation” includes mixtures of different formulations andreference to “the method of treatment” includes reference to equivalentsteps and methods known to those skilled in the art, and so forth.

[0018] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to describe and disclose specificinformation for which the reference was cited in connection with.

[0019] Red blood cells of primates contain high concentrations ofpotassium ion (K⁺). When primate blood is stored (as is the case withvirtually all blood obtained from blood banks), even low levels of lysisof the red blood cells generally result in high potassium ionconcentrations. This is due to release of potassium ion from inside thelysed primate red blood cells into the plasma surrounding the cells.Accordingly, the blood will be hyperkalemic when infused. The increasedpotassium level can be diffused if blood is infused into patients withsufficient circulating blood since the high potassium ion concentrationis diluted. However, the problem increases if primate blood istransfused into a maintenance solution of the type described in U.S.Pat. No. 4,924,442, which contains high concentrations of potassium. Thepotassium ion concentration in the transfused blood will not be dilutedto safe levels. As a result, cardiac insufficiency may and frequentlydoes occur. Hyperkalemia is also associated with tissue damage resultingfrom burns, accidents, surgery, chemotherapy, and other physicaltraumas. The prior art teaches that organ preservation at lowtemperatures requires the presence of high potassium ion concentrationsfor the maintenance of tissue integrity.

[0020] The solution according to the present invention contains asubphvsiological amount of potassium. Thus, the solution allows fordilution of the potassium ion concentration in stored transfused blood.As a result, high concentrations of potassium ion and potential cardiacarrhythmias and cardiac insufficiency caused thereby can be more easilycontrolled. The solution containing a subphysiological amount ofpotassium is also useful for purposes of blood substitution and lowtemperature maintenance of a subject. By “subphysiological amount ofpotassium” is meant between 0-5 mEq/l K⁺ (0-5 mM), preferably 2-3 mEq/1K⁺ (2-3 mM).

[0021] The solution of the present invention comprises a mixture ofmaterials which when placed in aqueous solution may be used to perfuse asubject in need thereof. While the materials may be provided as a drymixture to which water is added prior to heat sterilization, thesolution is preferrably provided in the form of a sterile aqueoussolution.

[0022] The solution of the present invention may be used as a singlesolution for all phases of procedures in which a subject's blood isremoved and replaced or a subject is cooled. Such phases includehemodilution or plasma extension at normal body temperatures, bloodreplacement and exchange at hypothermic body temperatures, bloodsubstitution at substantially hypothermic body temperatures, and subjectwarming. “Hypothermic body temperatures” are defined as 4-SoC belownormal body temperatures of 37-38° C. In other words, a hypothermiccondition may be considered to start at body temperatures of about32-35° C. “Substantially hypothermic body temperatures” are defined asbody temperatures just below the freezing point (−2° C.) to about 10° C.Therefore, the term “hypothermic body temperature” or “hypothermia” asused herein encompasses body temperatures of about −2 to 3° C. to about32-35° C.

[0023] The solution of the present invention does not include aconventional biological buffer. By “conventional buffer” is meant acompound which in solution, in vitro, maintains pH at a particularrange. By “conventional biological buffer” is meant a compound which ina cell-free system maintains pH in the biological range of 7-8. Examplesof conventional biological buffers includeN-2-Hydroxyethylpiperazine-N′-2-hydroxypropanesulfonic acid (HEPES),3-(N-Morpholino) propanesulfonic acid (MOPS), 2-([2-Hydroxy-1,1-bis(hydroxymethyl) ethyl] amino) ethanesulfonic acid (TES),3-[N-tris(Hydroxy-methyl)methylamino]-2-hydroxyethyl]-1-piperazinepropanesulfonicacid (EPPS), Tris[hydrolymethyl]-aminomethane (THAM), andTris[Hydroxylmethyl]methyl aminomethane (TRIS). Conventional biologicalbuffers function independently of normal biological processes, e.g., theconventional buffer is not metabolized in vivo, and are most potent incell-free systems.

[0024] The solution of the present invention uses normal biologicalcomponents to maintain in vivo biological pH, a concept termed a“dynamic buffering system”. The dynamic buffering system concept restson the discovery by the inventors that compounds with no intrinsicbuffering capacity in the biological range, such as lactate, capable ofbeing metabolized in vivo, act with other solution components tomaintain a biologically appropriate pH in an animal, even at hypothermictemperatures and at essentially bloodless conditions. The dynamicbuffering system of the present invention depends in part on oxygenationand removal of carbon dioxide (CO₂); and allows but does not requireadditional bicarbonate (NaHCO₃). The dynamic buffer of the invention hasno or substantially no ability to act as a buffer outside of abiological system, i.e., a dynamic buffer maintains pH in the biologicalrange in vivo but not in a cell free environment. A component of thedynamic buffering system of the invention include a carboxylic acid,salt or ester thereof. What is meant by a carboxylic acid, salt or esterthereof is a compound having the general structural formula RCOOX, whereR is an alkyl, alkenyl, or aryl, branched or straight chained,containing 1 to 30 carabons which carbons may be substituted, andpreferably one of the carbon chains that compose the carbon chain oflactate, acetate, citrate, pyruvate, or other biological metabolites;and X is hydrogen or sodium or other biologically compatible ionsubstituent which can attach at the oxygen position, or is a shortstraight or branched chain alkyl containing 1-4 carbons, e.g., —CH₃,—CH₂CH₃.

[0025] As shown in Table 1, a typical conventional buffer solution (25mM TRIS) that has an initial pH of about 7.7, and maintains a pH above7.2 with the addition of up to 0.12 mls of a 1.25 M HCl solution. Bycontrast, the pH of HLB solution (initial pH 7.7) drops below 7.2 withthe addition of about 0.01 ml of a 1.25 M HCl solution.

[0026] When the solution of the present invention is used as a bloodsubstitute at hypothermic temperatures, medical grade sterile NaHCO₃ isadded to the heat sterilized solution (HL solution). The solutioncontaining NaHCO₃ is called HLB solution. The buffering capacity of HLBsolution relative to a conventional biological buffer in a cell-freesystem is shown in Table 1. Under in vivo conditions with oxygenation,HLB solution is shown to maintain pH above 7.3 in temperatures rangingfrom 1.6-36.1° C. (Tables 2 and 3).

[0027] When the solution of the invention is used as a plasma extenderat normal body temperatures, in vivo pH is maintained in the biologicalrange without the addition of NaHCO₃.

[0028] The absence of a conventional biological buffer in the solutionof the invention confers the important medical advantage of allowing thesolution to be terminally heat sterilized. Generally, medical solutionsare preferred to be terminally heat sterilized prior to use in apatient. The term “terminally heat sterilized” or “heat sterilized” asused herein referes to the process involving heating a solution to 120°C. for 15 minutes under pressure, i.e., maintaining heat and pressureconditions for a period of time sufficient to kill all or substantiallyall bacteria and inactivate all or substantially all viruses thesolution. This procedure is normally performed in an autoclave, and isalso known as “autoclaving”. The purpose of heat sterilization is tokill possible infectious agents present in the solution. Infectiousagents are known to tolerate temperatures up to 100° C. It is generallyconsidered by the art that heating a solution under pressure to 120° C.for about 15 minutes is sufficient to insure sterility.

[0029] All transplant or blood substitute solutions of which theinventors are aware cannot tolerate terminal heat sterilization. It isknown that heat sterilizing a solution having a pH above 7.0 results insubstantial degradation of other solution components.

[0030] By contrast, the solution of the present invention is designed tobe heat sterilizable with minimal degradation of other solutioncomponents, such as sugar. Solution HL is heat sterilized prior to use.When it is desirable to add NaHCO₃ to form HLB solution, NaHCO₃ is addedas a commercially-available sterile 1 M solution to sterile HL solutoin.Generally, 5 mls of a 1 M NaHCO₃ solution is added per liter of HLsolution to form 1 l of HLB solution. However, more NaHCO₃ may be added.

[0031] The HLB solution of the present invention, or its bufferingorganic acids and salts, may also be used to sustain cultured tissuesand cells in vitro. The dynamic buffering system of the solutionmaintains cultured tissues and cells at the appropriate biological pH.We have shown that the addition of lactate and bicarbonate to culturedcells is sufficient to sustain normal cell growth and morphology.

[0032] The solution of the present invention includes an organiccarboxylic acid or salt thereof. The term “organic carboxylic acid orsalt thereof” includes any carboxylic acid or carboxylic acid derivativecapable of being metabolized by the mammal. Examples of carboxylic acidsand carboxylic acid salts suitable for use in the solution of thepresent invention include lactate and sodium lactate, citrate and sodiumcitrate, gluconate and sodium gluconate, pyruvate and sodium pyruvate,succinate and sodium succinate, and acetate and sodium acetate. In thefollowing Examples describing the use of HLB solution, sodium lactate isused. When metabolized in vivo, lactate helps maintain bicarbonatelevels, and thereby functions as a component of the dynamic bufferingsystem of the solution to maintain an in vivo biological pH.

[0033] For purposes of the further description of the invention, themixture according to the invention will be discussed as an aqueoussolution. From the following description of the invention, it isexpected that one ordinarily skilled in the art would be enabled toprovide the mixture as a dry mixture and make the adjustments to amountsof sodium chloride and organic salt of sodium as necessary toaccommodate the amounts of sodium chloride found in normal salinesolution, which may be used as a diluent for the dry mixture accordingto the invention.

[0034] The amount of organic salts of sodium is calculated in a mannerso as to consider the concentration of sodium ions present in thesubject's blood as well as the sodium chloride concentration of anysolution to which dry components are added. An amount is added so thatthe concentration of sodium ion obtained from the organic salt of sodiumis sufficient to bring the concentration of sodium ion in the solutionto a concentration about that of physiologically normal plasma.Therefore, when taking into account the amount or concentration ofsodium ion obtained from the organic salt of sodium and sodium chloride,the concentration of sodium ion in the solution is about theconcentration of sodium ion found in physiologically normal plasma.

[0035] The solution also includes a concentration of calcium, sodium andmagnesium ion which is within the range of normal physiologicalconcentrations of said ions in plasma. In general, the desiredconcentration of these ions is obtained from the dissolved chloridesalts of calcium, sodium and magnesium and in the case of sodium from adissolved organic salt of sodium which is also in solution.

[0036] The sodium ion concentration is preferably in a range from 70 mMto about 160 mM, and preferably in a range of about 130 to 150 mM.

[0037] The concentration of calcium ion is in a range of about 0.5 mM to4.0 mM, and preferably in a range of about 2.0 mM to 2.5 mM.

[0038] The concentration of magnesium ion is in a range of 0 to 10 mM,and preferably in a range of about 0.3 mM to 0.45 mM. It is importantnot to include excessive amounts of magnesium ion in the solutionaccording to the invention because high magnesium ion concentrationsnegatively affect the strength of cardiac contractile activity. In apreferred embodiment of the invention, the solution containssubphysiological amounts of Mg⁺⁺.

[0039] The concentration of chloride ion is in the range of 70 mM to 160mM, preferably in the range of 110-125 mM Cl⁻.

[0040] The solution also includes an amount of simple hexose sugar suchas glucose, fructose and galactose, of which glucose is preferred. Inthe preferred embodiment of the invention nutritive hexose sugars areused and a mixture of sugars can be used. In general, the concentrationof sugar is in a range between 2 mM and 10 mM with concentration ofglucose of 5 mm being preferred. At times, it is desirable to increasethe concentration of hexose sugar in order to lower fluid retention inthe tissues of a subject. Thus the range of hexose sugar may be expandedup to about 50 mM if necessary to prevent or limit edema in the subjectunder treatment.

[0041] The oncotic agent is comprised of molecules whose size issufficient to prevent their loss from the circulation by traversing thefenestrations of the capillary bed into the interstitial spaces of thetissues of the body. As a group, oncotic agents are exemplified by bloodplasma expanders.

[0042] Human serum albumin is a blood plasma protein used to expandplasma volume. Also known are polysaccharides, generally characterizedas glucan polymers which are used as blood plasma expanders. In general,it is preferred that the polysaccharide is non-antigenic.

[0043] Hetastarch (McGaw, Inc.) is an artificial colloid derived from awaxy starch composed almost entirely of amylopectin with hydroxyethylether groups introduced into the alpha (1 . . . 4) linked glucose units.The colloid properties of a 6% solution (wt/wt) of Hetastarchapproximates that of human serum albumin. Other polysaccharidederivatives may be suitable as oncotic agents in the solutions accordingto the invention including hydroxymethyl alpha (1 . . . 4) or (1 . . .6) polymers. Cyclodextrins are suitable oncotic agents.

[0044] D-glucose polymers may be used. For example, dextran, which isD-glucose linked predominantly in alpha (1 . . . 6) linkage, may be usedas the oncotic agent in the solution of the invention. Polysaccharidessuch as dextran in a molecular weight range of 30,000 to 50,000 daltons(D) are preferred. Most preferred is Dextran 40 having a molecularweight of about 40,000 D.

[0045] High molecular weight polysaccharides, such as Dextran 70, havinga molecular weight of about 70,000 D are generally less preferredbecause they increase the viscosity of the colloidal solution, therebyimpairing high flow rates. However, for some uses, high molecular weightdextran solutions are preferred in that they are more effective inpreventing tissue swelling due to their lower rates of leakage fromcapillaries. Thus, such high molecular weight dextran solutions areparticularly useful in the treatment of cerebral ischemia at hyperbaricoxygen tensions and in effectively managing cerebral oedema. In suchcircumstances, it may be desirable to use higher molecular weightpolysaccharide such as dextran in a molecular weight range of 50,000 to70,000 D.

[0046] When Dextran 40 is used in the solutions according to theinvention, about 8% Dextran 40 (wt/wt) or about 80 grams (g) per liter(1) of water is used. Molarity of the blood substitute according to theinvention will be in a range of about 290 to 330 milliMolar with amolarity of about 300 being preferred. Most preferred is a finalmolarity of about 298 mM.

[0047] The concentration of the polysaccharide is sufficient to achieve(when taken together with chloride salts of sodium, calcium andmagnesium, organic ion from the organic salt of sodium and hexose sugardiscussed above) colloid osmotic pressure approximating that of normalhuman serum, about 28 mm Hg.

[0048] The solution may be used as a circulating solution in conjunctionwith oxygen or hyperbaric oxygen at normal body temperatures, or with orwithout hyperbaric oxygen in subjects during procedures. The solutionmay also be used as a circulating solution in subjects during procedureswhen the subject's body temperature is reduced significantly below thesubject's normal temperature. When warm-blooded subjects are exposed tolow temperature conditions during surgical procedures and in cadaverorgan donation at low temperature, it is generally desirable to replacethe subject's blood with the cold circulating solution of the invention,or the solution circulated for a time, designed to perfuse and maintainthe subject and its organs intact during the procedure.

[0049] The solution of the present invention may be administeredintravenously or intraarterially to a euthermic subject which is placedin a pressurized atmosphere of increased oxygen concentration up to 100%oxygen or to such a subject undergoing a procedure during which thesubject's body temperature is reduced significantly below the subject'snormal temperature whether or not hyperbaric oxygen is used. While thesolution is being administered to and circulated through the subject,various agents such as cardioplegic agents may be administered eitherdirectly into the subject's circulatory system, administered directly tothe subject's myocardium, or added to the circulating solution of thepresent invention. These components are added to achieve desiredphysiological effects such as maintaining regular cardiac contractileactivity, stopping cardiac fibrillation or completely inhibitingcontractile activity of the myocardium or heart muscle.

[0050] Cardioplegic agents are materials that cause myocardialcontraction to cease and include anesthetics such as lidocaine, procaineand novocaine and monovalent cations such as potassium ion inconcentrations sufficient to achieve myocardial contractile inhibition.Concentrations of potassium ion sufficient to achieve this effect aregenerally in excess of 15 mM.

[0051] During revival of a subject (after a period of subnormaltemperature or cryogenic maintenance using the solution according to theinvention to maintain the subject) the subject may be reinfused with amixture of the solution according to the invention along with bloodretained from the subject or obtained from blood donors. As the subjectis warmed, whole blood is infused until the subject achieves anacceptable hematocrit, generally exceeding hematocrits of about 30%.When an acceptable hematocrit is achieved, perfusion is discontinued andthe subject is revived after closure of surgical wounds usingconventional procedures.

[0052] In general, the solution according to the invention isadministered using an intravenous line (when the subject is at normaltemperature) or to a chilled subject using a pumped circulating devicesuch as a centrifugal pump, roller pump, peristaltic pump or other knownand available circulatory pump. The circulating device is connected tothe subject via cannulae inserted surgically into appropriate veins andarteries. When the solution is administered to a chilled subject, it isgenerally administered via an arterial cannula and removed from thesubject via a venous cannula and discarded or stored.

[0053] The solution may be used in a variety of surgical settings andprocedures. It may be useful in delicate neurosurgery where clearsurgical fields are imperative and reduced central nervous systemactivity may be desirable and achieved by performing the procedure on apatient whose core temperature and/or cerebral temperature has beensubstantially reduced.

[0054] The solution may be used to maintain a subject (which has lost asignificant amount of blood, e.g. 20% to 98% of its blood) at normalbody temperatures in a pressurized environment at increased oxygenconcentration above atmospheric oxygen tension up to 100% oxygen. Thesubject is maintained in a high oxygen concentration until enough bloodcomponents can be synthesized by the subject to support life atatmospheric pressure and oxygen concentration. The solution according tothe invention may be used to maintain a subject at temperatures lowerthan normal body temperature and at a reduced rate of metabolism aftertraumatic life threatening injury until appropriate supportive orcorrective surgical procedures can be performed. In addition thesolution may be used to maintain a patient having a rare blood or tissuetype until an appropriate matching donor can be found and replacementblood units or other organ can be obtained.

[0055] Surprisingly it has been discovered that it is possible toreplace substantially all of a mammalian subject's circulating bloodwith the solution according to the invention and to maintain the subjectalive without reinfusing blood into the subject. Substantially all of amammalian subject's circulating blood is considered to be replaced whenthe subject's hematocrit drops below 10%. Hematocrit may be lower than10% if O₂ is provided to the subject, or substantially lower than 10% ina hyperbaric O₂ chamber. The solution according to the invention can ofcourse be used to maintain a subject having a hematocrit in excess of10%.

[0056] The procedure for replacing substantially all of a mammaliansubject's circulating blood may be carried out with the mammaliansubject's body temperature being maintained at its substantially normaltemperature. In addition the procedure may be carried out with coolingof the subject and reduction of the mammalian subject's body temperaturebelow that of its normal temperature. Cooling may be accomplished bychilling the subject in an ice bath, ice-salt slurry, or coolingblanket. The subject may be further cooled by chilling the solutionaccording to the invention prior to perfusing the subject with thesolution.

[0057] In the procedure according to the invention for replacingsubstantially all of a mammalian subject's circulating blood, it ispreferred that the subject is chilled and perfused with the solution,using an arterial catheter to deliver the solution to the subject'scirculatory system and a venous catheter to remove blood and theperfusate from the subject. Substantially all of the subject'scirculating blood is removed in this manner as determined by measurementof the hematocrit of the effluent from the venous catheter. Whensubstantially all of the subject's circulating blood is removed,perfusion is stopped.

[0058] In addition, the procedure for replacing substantially all of thesubject's blood may be carried out with the aid of hyperbaric O₂. Thesubject is placed in a hyperbaric chamber pressurized with oxygen atconcentrations exceeding 20%, preferably 100% oxygen. The pressure ofthe hyperbaric chamber is maintained during most of the procedure in arange between 0.5 pounds per square inch over atmospheric pressure topressures up to about twice atmospheric pressure. In one embodiment, theprocedure is performed with the subject in a hyperbaric chamber athyperbaric pressures of about 0.07 to about 2 atmospheres over ambientpressure (0.5-30 pounds per square inch [psi]) with 100% oxygen. Ifnecessary, the pressure of the hyperbaric chamber may be reduced toatmospheric pressure during wound closure. The subject is subsequentlymaintained at hyperbaric pressure at high oxygen concentration. Thepressure is gradually reduced to a lower pressure but one stillhyperbaric. Preferably the pressure is maintained below 10 psi to about5 psi for a number of hours to several days. Subsequently, the pressureis again gradually lowered below 1 psi and preferably to about 0.5 psiand is maintained at this pressure for an additional period of time upto a day or more.

[0059] The solution may also be used to maintain the physiologicalintegrity of an organ donor subject immediately after the occurrence ofbrain death. The subject can be chilled, the subject's blood removed andreplaced with a circulating solution maintained below 37° C., or whilecirculating cold solution according to the invention. Through this useof the solution, ischemia of vital organs can be minimized. Bycirculating cold solution according to the invention through thesubject's circulatory system at low temperature with or without placingthe subject in a hyperbaric oxygen chamber, vital organs can bemaintained for longer periods of time, thus maximizing the number oforgans that can be effectively used from one donor for potentialtransplant recipients.

[0060] In another aspect of the invention, it has been discovered thatby using certain adducts, particularly propanediol and highconcentration glucose to augment the solution, it may be possible toreduce the temperature of donor organs, and in particular donor hearts,below the freezing point of water (0° C.) and recover them from freezingin a useful state, i.e. a state capable of maintaining coordinatedcardiac contraction. Furthermore by using the solution according to theinvention with such adducts, it has been possible to reduce thetemperature of intact mammalian donor subjects below the freezing pointof water (0° C.) and restore them from freezing in a state capable ofmaintaining coordinated cardiac contraction. Other organ systems arealso believed to be maintained with a high degree of biologicalintegrity, i.e. in a physiological state capable of maintaining life.

[0061] The adducts to the solution include low molecular weightaliphatic polyalcohols. Diols, exemplified by ethylenediol, propanediol,and butanediol are preferred. Of these diols propanediol is particularlypreferred. Other polyalcohols that may be suitable as adducts for lowtemperature, sub-zero OC preservation of organ and organ donor subjectsare low molecular weight polyethylene glycol. It is preferred in thisaspect of the invention that the adduct is added to the solution to afinal concentration in a range between about 0.2 Molar to 1 Molar. Withrespect to propanediol, in particular a range of 0.2M to 0.6M ispreferred. A concentration of about 0.4M propanediol is most preferred.1,2 propanediol is preferred as the adduct to the solution used for lowtemperature organ and donor preservation according to the invention,although 1,3 propanediol may be used.

[0062] The glucose concentration in the solution useful for sub-zero 0°C. preservation of organ and organ donor subjects ranges between about0.6M to about 1.4M. A concentration of about 1M glucose is preferred.

[0063] Another adduct that is useful in the solution for low temperatureand sub-zero ° C. preservation of organ and organ donor tissues istrimethylamine oxide (TMAO). TMAO may be added to the solution describedimmediately above to a final concentration in a range between 0.2M and7M. The solution including TMAO when perfused into a subject leads toimproved biological integrity of the subject's tissues as evidenced bysuperior anatomical preservation of the tissues.

[0064] The following Examples are intended to illustrate the inventionand its use, and are not intended by the inventors to be limiting of theinvention.

EXAMPLES

[0065] The following example is put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to carry out the synthesis of the invention and is not intended tolimit the scope of what the inventors regard as their invention. Effortshave been made to ensure accuracy with respect to numbers used (e.g.,amounts, temperature, etc.), but some experimental error and deviationshould be accounted for. Unless indicated otherwise, parts are parts byweight, molecular weight is weight average molecular weight, temperatureis in degrees Centigrade, and pressure is at or near atmospheric.

Example 1

[0066] Solution Preparation.

[0067] Preparation of 10 L of Solution A.

[0068] Into an appropriate container, add 80 g/L (or 800 g for 10liters) of pyrogen-free Dextran 40 (Pharmachem or Pharmacia). Adddeionized water, bringing the volume up to 6-9 liters. Dissolve theDextran 40 completely by shaking. The following components may be addedin any order, dissolving each completely before the addition of thenext. The following reagents may be obtained from chemical supplyhouses; in this example the listed reagents were obtained from Sigma:NaCl (5.2 g/L), CaCl₂ (0.29 g/L), MgCl₂ (0.40 g/L), glucose (0.9 g/L),Tris (3.03 g/L), and sodium gluconate (6.54 g/L).

[0069] Next, the solution is brought to pH 7.80 at room temperature bythe dropwise addition of 0.25M HCl while shaking and monitoring with apH meter. The solution is then brought to its final desired volume (i.e.10 liters) by the addition of more deionized water.

[0070] Finally, the solution is pumped through a 0.2μ filter (Gelman,Whatman, or ideally Pall filter units can be used) into sterilecontainers or bags. The bottled and capped solution is stored on iceuntil used.

[0071] The solution may then be prepared as a sterile dry powder incontainers suitable for preparation of sterile IV solutions after freezedrying under appropriate conditions.

[0072] Preparation of Solution HL.

[0073] To prepare 50 liters of solution L (BioTime Hextend™-lactate),3.0 kg of high molecular weight Hetastarch (HES) is added to 25 litersof water. Sufficient NaCl is added to bring the final NaCl concentrationto 6.72 g/l. The solution is stirred until both the HES and NaCl aredissolved. The solution may be heated to 50° C. if necessary. The totalvolume is brought to 45 liters and the following components are addedand mixed until completely dissolved: CaCl₂.2H₂O 18.5 g; MgCl₂.6H₂O 4.5g; KCl 11.0 g; glucose 45.0 g; and sodium lactate 4.03 ml of a 60t(wt/wt) solution. The solution is brought up to a volume of 50 liters.The solution is filtered to remove undissolved material and placed inautoclavable containers and heated in an autoclave to a temperature of120° C. for 15 minutes.

[0074] Solution HLB.

[0075] To each heat sterilized liter or HL solution is added 5 ml of asterile 1 M solution of NaHCO₃, medical grade, forming HLB solution(BioTime Hextend™-lactate-bicarbonate).

[0076] Solution L.

[0077] Solution L is prepared as described for HL solution above withoutthe addition of Hetastarch (HES).

Example 2

[0078] Hamster Revived after 1 Hour of Ice-cold Blood-substitution.

[0079] A 41 g female hamster (Mesocricetus auratus), approximately 1month old, was injected i.m. with 0.04 ml of Vetalar, a 100 mg/mlsolution of the anesthetic ketamine. The animal was packed in crushedice and chilled until its rectal temperature was 10° C. The animal wasremoved from the crushed ice and placed ventral side up on acustom-designed stage positioned so that specific portions of the animalcould be observed through a stereo-microscope during surgery. Its limbswere secured, and the animal was instrumented with EKG leads and arectal telethermometer probe.

[0080] An incision was made in the right groin region, and the rightfemoral vein, and then the right femoral artery, were cannulated usingspecially designed micro-cannulas filled with solution A. Aftercannulation, 0.02 ml of heparin (1000 U/ml) in solution A was injectedinto the animal through the venous cannula, which was then capped.

[0081] After the right femoral arterial cannulation, the cannula wasconnected to a luer-tipped segment of sterile plastic tubing which wasconnected to a stopcock mounted on the surgical stage. The stopcock wasconnected to another tubing segment which was in turn connected to awider, thicker, and more compliant tubing segment passed through thehead of a roller pump. The end of this wider tubing segment contained atube for drawing up fluid from a reservoir. This tube for drawing upfluid from a reservoir termed a “pick-up” herein was fashioned from theluer end of an 18 gauge hypodermic needle. This “pick-up” was coveredwith blood filter material which was secured by a small rubber “O” ring.The “pick-up” was inserted into a reservoir of solution A contained by acentrifuge tube immersed in crushed ice. 0.06 ml of 1M KCl was added tothe solution (15 ml), yielding a molar concentration of about 4 mM KCl.The line was closed using the stopcock to prevent back-bleeding into thearterial cannula.

[0082] The hamster was surrounded with crushed ice, and chilled to 4° C.Then 0.2 ml of 1M KCl was injected into the stopcock, which was openedto allow the injected solution to flow into the line connecting to thearterial cannula, and from there, into the animal's femoral artery. Thehamster's heart arrested. The animal was allowed to cool further, andwas perfused through the arterial cannula with 8 ml of solution A 4 mMKCl. Effluent, containing most of the hamster's blood, was collectedfrom the venous cannula. After the hematocrit dropped below 5, theroller pump was turned off for 67 minutes.

[0083] The hamster was then perfused through the arterial cannula with 8ml of solution A without KCl, followed by 8 ml of heparinized bloodtaken from other hamsters by cardiac puncture. An equal amount ofeffluent was collected from the venous cannula. After the hematocritexceeded 40%, perfusion with whole blood was ended, and the cannulasremoved.

[0084] The hamster was warmed with a desk lamp, until it became reactiveto stimuli. The cannulas were removed, open blood vessels ligated, andincisions closed. Further rewarming continued. The animal fullyrecovered, and continued to live for weeks following the experiment.

Example 3

[0085] Cardiac Preservation After Sub-zero Storage.

[0086] A fasted (overnight) female hamster, 40 grams, was injected,i.m., with 0.02 ml of Ketamine anesthetic (100 mg/ml). The hamster wasimmersed in crushed ice until its body temperature lowered to +14° C. Itwas then placed on a surgical stage and instrumented with EKG leads anda rectal temperature probe. The carotid artery and jugular vein wereexposed surgically while the animals body temperature was maintainedbetween 10-14° C. and cannulas were inserted into the artery and vein.The arterial cannula was attached to tubing connected to a peristalticpump. The tubing was filled with solution A, containing in addition 20mM KCl. The venous cannula was capped until the animal's bodytemperature was lowered to 5° C. using crushed ice and atemperature-controlled stage set at −1.0° C.

[0087] The animal stopped breathing on its own when its body temperaturefell below 10° C. Respiration with 100% O₂ was initiated. At 5° C., thevenous cannula cap was removed and 3.5 ml of solution A was pumped intothe artery at a flow rate of about 0.3 ml/minute. Afterwards, 4.5 ml ofa cryoprotective solution composed of solution A and in addition 4 mMKCl, 1.0M glucose, 4% propanediol (i.e. 1.8 g glucose +0.4 g propanediolper 10 ml solution) was infused. During perfusion, the venous effluentwas collected. The animal's temperature was lowered gradually to 0° C.during perfusion. Respiration was discontinued 5 minutes following theonset of perfusion. At this time, more than 30% of the subject's bloodvolume had been removed. The heart continued beating until it eventuallystopped. Following perfusion with the cryoprotective solution describedin the preceding paragraph, the animal was placed in a sub-0° C. NaClslush (0.6M) solution which was placed in a freezer overnight.

[0088] The freezer temperature was kept at an average of −5° C. Fifteenminutes after the animal was placed in the freezer, its rectaltemperature lowered from 0 to −1.0° C. The animal's rectal temperature12 hours later was −2.5° C. The animal was then warmed to a temperatureof about 2.5° C. in a Quasar commercial kitchen microwave oven using 7second pulses with the setting on warm. The pulses were generated 1minute apart. Eighteen pulses were needed to thaw the animal.

[0089] The animal was again placed on the surgical stage andinstrumented with EKG leads and a rectal telethermometer probe. Threeand one half ml of solution A was perfused into the carotid artery at aflow rate of approximately 0.2 ml/min. The animal's body temperature wasmaintained below 5° C. The hamster was then perfused with whole blood,and gradually warmed.

[0090] After 2 ml of blood had been infused, and the animal'stemperature had climbed to 13° C., rhythmic EKG signals were detected.With continued perfusion and warming, the amplitude of the signalsbecame greater, and they increased in frequency. After 5.5 ml of bloodhad been infused, and the animal's temperature had reached 25° C., thechest of the animal was opened and its heart was observed to beatcontinuously.

Example 4

[0091] Synthetic Solution Substitutes for Blood in a Hvperbaric Chamber.

[0092] A 40 g hamster, previously fasted overnight, was injected with0.03 ml Ketamine (100 mg/ml) i.m. The hamster was placed in crushed ice,until its body temperature fell below 15° C. The hamster was removedfrom crushed ice, and placed ventral side up on a temperature-controlledstage positioned for microsurgery below a stereo-microscope. Thehamster's temperature was maintained between 12-15° C.

[0093] Following an incision in the right groin area, the right femoralvein and artery were exposed. The femoral vein was cannulated, 0.1 ml ofheparin (1000 u/ml) was injected, and the cannula was capped to preventbleeding.

[0094] The right femoral artery was then cannulated, and the cannula wasbriefly attached to tubing filled with solution A. The tubing wasthreaded through the head of a peristaltic pump. A small volume of thesolution (approximately 0.3 ml) was infused to keep the arterial cannulavoid of blood. Both the venous and arterial cannulas were secured to theanimal with surgical suture.

[0095] The arterial cannula was capped and the animal was moved onto thestage in a hyperbaric oxygen (HBO) chamber. A temperature probe wasinserted into the rectum.

[0096] The arterial cannula was attached to tubing which passed througha peristaltic pump and into a reservoir. The tubing and reservoir werefilled with solution A containing 4 mM KCl.

[0097] The cap was removed from the venous cannula, and the HBO chamberwas closed and pressurized. The peristaltic pump was turned on, and theanimal perfused with solution, which replaced most of its blood. Thisblood was allowed to drain from the animal as a venous effluent. Thefinal chamber pressure was 1.5 atm over ambient pressure, which was keptconstant. The flow rate of solution into the animal was about 0.3ml/min. The hamster was maintained between 14-16° C. using thetemperature-controlled stage on which the hamster was positioned in theHBO chamber.

[0098] Cardiac activity and breathing were maintained throughout thisperiod during the perfusion. After 15 ml of solution A containing inaddition 4 mM KCl was perfused into the hamster replacing the blood, thechamber was gradually depressurized.

[0099] The chamber was then opened, and a hematocrit sample was taken.The hematocrit was 5%. The venous and arterial cannulas were capped andthe chamber closed and pressurized to 1.5 atm over ambient pressure.

[0100] The animal continued to breathe on its own in the chamber for 4hours after the removal of its blood. After this time, the chamber wasdepressurized gradually. Concomitantly, the animal was cooled to 12° C.The chamber was opened, and the animal was moved to another surgicalstage. Ice was placed on the animal, and whole blood was perfused intothe animal at a flow rate of 0.2 ml/min, as solution was allowed todrain as venous effluent.

[0101] After 1 ml of blood was infused, the ice was removed. Thehamster's body temperature was at 4° C. The animal was then permitted towarm gradually as the hematocrit was raised by continuous bloodinfusion.

[0102] Artificial respiration was initiated after 1 ml of blood was putback in. The animal's heart never stopped beating rhythmically. At 21°C., the animal was breathing steadily on its own. Artificial respirationwas discontinued and warming and blood infusion continued until theanimal's temperature reached 25° C. The hematocrit was measured to be40%. Perfusion was discontinued, the cannulas removed, blood vesselsligated and surgical incisions closed.

[0103] One hour following the procedure, the animal was very active andalert. Four hours after the experiment, the animal was eating anddrinking. At 24 hours after the completion of the above-describedprocedure, it appeared completely normal with respect to posture andbehavior, and continued to live for weeks after the experiment.

Example 5

[0104] Ice-cold Blood Substitution of a Hamster.

[0105] A 46 g hamster, approx. 1 month old, was injected i.m. with 0.02ml Vetalar, a 100 mg/ml solution of ketamine. The animal was surroundedby crushed ice until its rectal temperature was about 12° C. The animalwas then removed from the crushed ice and placed ventral side up on anoperating stage designed to keep the animal cold, which is under astereo-microscope. Its limbs were secured, and the animal wasinstrumented with EKC leads and a rectal telethermometer probe.

[0106] An incision was made in the right groin region. A cannula wasplaced in the right femoral vein, and 0.02 ml of heparin solution (250U/ml) was injected into the animal through the cannula which was thencapped. Then the right femoral artery was cannulated. The cannula wasconnected to a luer-tipped segment of plastic tubing, and the tubing waspassed through a peristaltic roller pump and into a reservoir containingsolution A containing 0.05 M glucose. At the end of the tubing wasinserted an 18G hypodermic needle to which a mesh blood filter materialwas secured at the hub by a rubber “O” ring. The pump was turned on, andfluid in the reservoir was pumped through the tubing into the femoralartery of the animal. When the animal's temperature fell below ₉₀C,ventilation (at 20 breaths/minute) was initiated using 100% oxygen. Theanimal was cooled further to a rectal temperature of 4° C., and 0.1 mlof 0.2M KCl was injected into the 24 G angiocath which was inserted inthe femoral vein. This injection arrested the heart, and EKG signalsceased. The pump was turned on, and solution A was perfused into theartery at approximately 0.2 ml/min while venous effluent was collected.During the perfusion the animal's temperature dropped to near 1° C.After 4 ml of solution was perfused into the animal, the pump was turnedoff and the animal was kept surrounded by crushed ice in circulatoryarrest for 2 hours. Then the animal was perfused with approximately 7 mlof whole blood (which was collected from other hamster blood donors)while the animal was gradually warmed using a desklamp. During theperfusion venous effluent was collected. The same volume pumped into theartery is collected as venous effluent. At 10° C., after the animalremained in cardiac arrest for 3 hours and 11 minutes, heart beats werefirst observed upon monitoring EKG signals. Ventilation (6breaths/minute) of the animal was then initiated using 100% oxygen. Asthe animal was further warmed and heart beats became stronger andfaster, this rate was increased to about 15 breaths/minute. When theanimal's temperature was above 28° C. the animal began to breathe on itsown and became responsive. Perfusion was discontinued (the hematocritreading 44%) and cannulas were removed and surgical wounds closed. Thishamster remained alive in apparently normal health for many weeks afterthe experiment.

Example 6

[0107] Recovery of Heart Beat in an Ice-cold Hamster.

[0108] A fasted (overnight) female hamster, 45 grams, was injected i.m.with 0.03 ml ketamine anesthetic (100 mg/ml). The hamster was immersedin crushed ice until its body temperature lowered to about 14° C. Theanimal was then placed on a surgical platform and instrumented with EKGleads and a rectal temperature probe. The carotid artery and jugularvein were exposed surgically using a stereo microscope. The animal'sbody temperature was maintained between 10-14° C. Cannulas were insertedinto the carotid artery and jugular vein. The arterial cannula wasconnected to tubing which passed through a peristaltic pump into areservoir containing cryoprotective solution composed of solution Acontaining, in addition, 11 mM KCl, 1.0 M glucose and 4% propanediol.The venous cannula was initially capped until the animal's bodytemperature was lowered to 5° C. using crushed ice and a temperatureregulated platform set near —1.0° C.

[0109] The animal stopped breathing on its own as the body temperaturefell below 10° C. At this time the animal was ventilated at about 15breaths per minute with 100% oxygen. When the animal's temperature fellto 5° C., the venous cap was removed and the pump was turned on at aflow rate of about 0.20 ml/minute. The animal's heart stopped beating 21minutes later, and ventilation was discontinued 5 minutes after theonset of perfusion. During the perfusion blood was collected as venouseffluent. Approximately 4 ml of the cryoprotective solution A wasinfused into the animal. Then the animal was surrounded by a salt-iceslurry whose temperature was −2.0° C. The container that held the slurryand animal was placed inside a temperature bath set at −5.0° C. Theanimal's rectal temperature gradually lowered to −3.4° C. in the morning(18 hours after the animal was put in the cooling bath). The containerwas removed from the cooling bath. The slurry was frozen solid. It wasmelted using ice-cold water. Upon removing the “slurry” the animal feltfrozen. The animal was then placed in a kitchen microwave oven. The ovenwas set on warm for 7 seconds. The animal was exposed to about 20, 7second heating cycles over a 20 minute period. This thawed the animaland raised its rectal temperature to about 2° C.

[0110] The animal was again placed on the surgical platform, and theanimal was infused into the carotid artery with solution A. Thecryoprotective solution was collected as venous effluent. About 3 ml ofsolution A was perfused into the animal at a flow rate of 0.15ml/minute. Blood which was collected from hamster blood donors was thenperfused in at the same flow rate. After 2 ml of blood was perfused intothe artery of the hamster, the hamster was warmed slowly using a desklamp. As blood perfusion and warming continued, the animal's temperaturerose above 15° C. and strong rhythmic EKG signals were recorded. Uponsurgical thoracotomy actual heartbeats could be observed.

Example 7

[0111] Synthetic Solutions as a substitute for Blood in a HyperbaricOxygen Chamber.

[0112] A 43 gram female hamster (fasted overnight) was injected, i.m.,with 0.02 ml of ketamine (100 mg/ml). The hamster was placed in crushedice until its body temperature fell to about 14° C. The hamster was thenplaced ventral side up on a temperature-controlled stage positioned formicrosurgery below a stereo-microscope. The hamster's temperature wasmaintained between 12-15° C. Following an incision in the right groinarea, the right femoral vein and artery were exposed. The femoral veinwas cannulated, 0.1 ml of heparin (250 u/ml) was injected, and thecannula was capped to prevent bleeding. The right femoral artery wasthen cannulated, and the cannula was attached to tubing passed through aperistaltic pump and into a reservoir filled with solution A. A smallvolume of the solution (i.e. 0.2 ml) was infused to keep the arterialline void of blood. Both the venous and arterial cannulas are secured tothe animal. The arterial cannula was capped, and the animal wastransferred onto the temperature-regulated stage of a hyperbaric oxygen(HBO) chamber. The animal's temperature measured rectally was maintainedbetween 13-18° C. The purpose of maintaining the hamster in thattemperature range was to keep the animal's activity low while ensuringthe animal was breathing on its own and reflexively responsive tostimuli.

[0113] The arterial cannula was connected to tubing that passed outsidethe chamber through a peristaltic pump and into a reservoir (inside thechamber) which contained solution A and 2.5 mM KCl. The cap was removedfrom the venous cannula, and the pump was turned on at a flow rate ofabout 0.2 ml/min. As the solution was perfused into the animal, venouseffluent (blood) was collected. The chamber was quickly closed andgradually pressurized to 20-24 psi (100% oxygen). After about 1 hour ofperfusion under pressure the chamber was gradually depressurized over aperiod of about 1 hour. Then perfusion was discontinued. A total ofabout 13 ml of solution was perfused into the animal. The cannulas werecapped after a sample of venous effluent was taken to determine thehematocrit. The animal was placed again on a surgical platform, and thecannulas were pulled out and wounds tied. The animal showed some veryminimal reflex activity during this time although the animal had littleblood and was breathing room air. The animal was quickly placed in a boxinside the chamber which was pressurized gradually to about 20 psi. Inthe chamber was placed food and water for the hamster. A heat lamp wasused to warm the chamber and the animal. The pressure in the chamber wasgradually lowered (over a 1 hour period) to 5 psi. The animal's activityincreased over the one hour period until it became quite active. Theanimal was maintained in the chamber for about 16 hours at 5 psi. Thepressure was then gradually lowered to 0.5 psi (100% oxygen) andmaintained at that pressure 24 hours. Then the animal was taken out ofthe chamber and was placed in a normal cage. The animal continued toappear completely normal many weeks following the experiment.

Example 8

[0114] Use of Solution a Augmented with Potassium

[0115] Chloride to Blood Substitute Primates In this example an 8 kg.juvenile male baboon of the species PaTio anubis was injected i.m. with60 mg of ketamine. A 22 gauge×1¼ in. catheter was inserted in the rightcephalic vein, and 3 ml of 2.5% pentothal was injected i.v. The animalwas then fitted with an endotracheal tube, placed on a surgical table,and ventilated with a 0.7-2.5% mixture of Flether in 100% O₂, titratedto the animal's activity. The eyes were coated with lacrylube forprotection.

[0116] The ventilator was set at 18 breaths per minute (bpm), its strokevolume was 240 ml, and the inspiratory/expiratory ratio was 37%. Airwaypressure was maintained at approximately 10 mm Hg, and the volumedelivered with each respiration was checked by examining the airwaypressure trace on a CRT or strip-chart recorder. Airway pressure wasmonitored on-line by computer.

[0117] The animal was shaved, and Ringer's lactate drip was initiatedi.v. at a flow rate of 1-3 ml/minute with the rate titrated to theanimal's arterial blood pressure. Terramycin was administered.

[0118] The extracorporeal circuit consisted of a blood oxygenator, bloodreservoir and pump and was constructed with a secondary in-line heatexchanger added as close to the animal as possible. It was furtherequipped with an external ice water reservoir. The ice-water reservoirhad a pump to supply the oxygenator's built-in heat exchanger, as wellas the secondary heat exchanger with circulating ice water. All tubingin contact with blood or blood substitute was sterile. The oxygenatorreservoir and circuit was filled with 2 liters of solution A.

[0119] KCl (4 ml of 2.0 M) was added to the 2 liters of solution A in anoxygenator reservoir and bypass circuit, yielding a KCl concentration of4 mM. A SF NIH catheter for monitoring arterial pressure was introducedinto the left brachial artery. To it was attached a 3-way stop-cock (toallow arterial blood sampling every 10-60 minutes throughout the entireprocedure). Blood gases, pH, K+ and hematocrit were measured in eachsample, and in some cases, electrolytes, and enzymes as well. Thecatheter was attached to a pressure transducer. The transducer wasconnected to a computer to monitor central arterial pressure (CAP).Other temperature and pressure parameters were also measured on-line bythe same computer.

[0120] A 6F NIH catheter was inserted into a distal branch of the leftbrachial vein to allow computerized monitoring of central venouspressure (CVP). A thoracotomy was performed, and a 6 F coronary catheterwas inserted into the left atrium to monitor left atrial pressure.

[0121] A 10 F arterial cannula was placed in the left femoral artery anda 16F venous cannula was placed in the left femoral vein. Methylprednisolone (80 mg) was introduced i.v. An esophageal tube wasinserted, and 3 ml of Maalox was administered. The esophageal tube wasfitted with a thermistor probe for recording deep esophagealtemperature.

[0122] Due to the extensive surgical procedures, the baboon spent aboutfive hours on anesthetic. After the EKG leads were in place, the animalwas put in a netted sling and lowered into an insulated ice chest. Itwas then immersed in crushed ice. After 1 hour and 6 minutes of chillingin crushed ice, body temperature sank to 23° C. Nipride (25 mg sodiumnitroprusside in 500 ml of 5% aqueous dextrose) infusion was begun at arate of 6 ml/hr. The animal was placed on bypass 17 minutes later, whenthe temperature had declined to 21° C.

[0123] At that time, 200 ml of whole blood were removed from the baboonas venous effluent. The clamps were released which isolated the monkey'scirculation from the bypass circuit, and 2 liters of solution A, towhich were added 2 ml of 2M KCl (final concentration 2 mM KCL), wereallowed to blood-substitute the animal. Following this, its heart wasarrested by the i.v. administration of 15 ml of 2M KCl.

[0124] A blood-blood-substitute mixture was continuously removed as avenous effluent until 4 liters of solution A (to which 22 ml of 2M KClhad been added) replaced the circulating solution. After 50 minutes ofchilled blood substitution, the primate's temperature had declined to 3°C. Flow through the animal appeared good, and there was little tendencyfor the pulmonary arterial wedge pressure to elevate along withperfusion of the femoral artery. The cause of this increased flow, andrelatively rapid pace of temperature decline, may be related to the useof nitroprusside, and also the relatively sparing use of anestheticsduring chilling, which resulted in the animal being somewhat more activeas it was cooled.

[0125] Following blood-substitution, the animal was placed oncirculatory standstill for one hour and 40 minutes. At the end of thestandstill period, 2 liters of ice-cold solution A was added to thecircuit, replacing 2 liters removed as venous effluent. The minimum bodytemperature recorded was 2.8° C. Rewarming was then begun. After 13minutes of warming, the animal's body temperature reached 10° C., and800 ml of a 1:3 mixture of blood and blood-substitute, followed by 450ml of a 1:1 mixture, and finally, approximately 1 liter of whole bloodwas added to the circuit, replacing solution A.

[0126] Immediately after blood was introduced into the animal, heartbeatwas detected. Over the next hour and 22 minutes, 40 ml of NaHCO₃, wereintroduced i.v. Mechanical ventilation was begun, and a dopamine drip(200 mg in 250 ml) was administered at 30 ml/hr. CaCl₂ (50 mg) was alsoinjected i.v. Approximately one hour later, when the body temperatureclimbed to near normal, the animal was taken off bypass and placed on awhole blood drip. The animal's blood gases and blood pressuresstabilized in the normal range.

[0127] One hour later, the cannulas were removed. Since the animal hadbeen catheterized following a thoracotomy, it was decided that the longterm post surgical management of the animal would not be attempted, dueto the behavioral problems of restraining an untamed baboon whiletreating potential chest infections. When ventilation was discontinuedafter another hour, the animal displayed agonal movements and went intocardiac arrest. As the monkey's blood pressures and blood gases hadstabilized, it is clear that the animal had the potential to surviveafter being blood-substituted below 10° C. (deep esophageal temperature)for 2 hours and 30 minutes.

Example 9

[0128] Use of Solution A Without Aucmentation in Blood Substitution ofPrimates

[0129] In this example an 8 kg juvenile male baboon of the species Papioanubis was chilled and blood-substituted below 10° C. for 1 hour and 22minutes. Prior to chilling and blood replacement, a 4F 60 cm Swan-Ganzarrow wedge catheter was placed in the pulmonary artery via the rightfemoral vein. This permitted measurement of the pulmonary arterial wedgepressure without performing a thoracotomy.

[0130] Keeping the animal anesthetically light, and using nitroprussidewhen the temperature fell to 28° C., improved flow through the bypasscircuit. Although the entire procedure went smoothly, an i.v. injectionof 50 mg calcium chloride after citrated blood was introduced duringwarming caused massive clot formation and termination of the experiment.At that time there was no heparin in the cardiovascular system.

[0131] Procedure.

[0132] The baboon was injected i.m. with 70 mg of ketamine. A 22gauge×1¼ in. catheter was inserted in the left cephalic vein, and 3 mlof 2.5% pentothal was injected i.v. The ape was then fitted with anendotracheal tube and moved to the x-ray room. It was placed on an x-raytable, and ventilated with a 1% mixture of isofluorane (Flether) in 100%O₂, and a 4F 60 cm arrow wedge catheter was implanted in the pulmonaryartery through the right femoral vein.

[0133] The ventilator was set at 20 bpm, its stroke volume was 200 ml,and the inspiratory/expiratory ratio was 37%. Airway pressure wasmaintained at approximately 10 mm Hg, and the volume delivered with eachrespiration was checked by examining the airway pressure trace on a CRTor strip-chart recorder. Airway pressure was monitored on-line bycomputer.

[0134] The animal was shaved, and a 1-3 ml/minute Ringer's lactate dripwas initiated i.v., with its rate titrated to the animal's arterialblood pressure.

[0135] The extracorporeal circuit was as described in the previousExample. The oxygenator reservoir and circuit was filled with 2 litersof solution A.

[0136] A 20 gauge hydromere catheter was placed in the right femoralvein to allow computerized monitoring of central venous pressure (CVP).A 3-way stopcock was placed in-line to allow sampling. A 20 gaugehydromere catheter for monitoring arterial pressure was introduced intothe right brachial artery. To it was attached a 3-way stop-cock (toallow arterial blood sampling every 10-60 minutes throughout the entireprocedure). Blood gases, pH, K+ and hematocrit were measured in eachsample, and in some cases, electrolytes, and enzymes as well. Thecatheter was attached to a pressure transducer. The transducer wasconnected to a computer to monitor central arterial pressure (CAP).Other temperature and pressure parameters were also measured on-line bythe same computer.

[0137] A 14F venous cannula was placed in the left femoral vein, and a10 F arterial cannula was placed in the left femoral artery. After thevenous cannula was implanted, 2.6 ml of heparin was injected i.v. Anesophageal tube was inserted, and 3 ml of Maalox was administered. Theesophageal tube was fitted with a thermistor probe for recording deepesophageal temperature. Methyl prednisolone (80 mg) was introduced i.v.The eyes were coated with lacrylube for protection. As the animal wasanesthetically light, 1 ml of pentothal was administered i.v.

[0138] The EKG leads were in place, the animal was put in a netted slingand lowered into an insulated ice chest. It was then immersed in crushedice. After 29 minutes of chilling in crushed ice, body temperature sankto 28° C. The animal was kept anesthetically light, Flether being turnedoff as the temperature dropped below 30° C. Nipride (sodiumnitroprusside—25 mg in 500 ml of 5% aqueous dextrose) infusion was begunat a rate of 20 ml/hr and then increased to 40 ml/hr. Over the next 20minutes, the Nipride drip was turned on and off sporadically, as theblood pressure and temperature fell. It was finally turned off when theanimal was placed on bypass 27 minutes later and the temperature haddeclined to 23° C. At that time, the clamps were released which isolatedthe ape's circulation from the bypass circuit, 2 liters of solution Awere allowed to blood-substitute the animal, and whole and diluted bloodwere removed as venous effluent, and saved for revival. Following this,its heart was arrested by the i.v. administration of 10 ml of 2M KCl.

[0139] A blood-blood-substitute mixture was continuously removed as avenous effluent until 4 liters of solution A replaced the circulatingsolution. After 39 minutes of chilled blood substitution, the primate'stemperature had declined below 4° C. Flow through the animal was rapid.The pressure in the pulmonary circulation, which was readily measured,indicated that the circulation was good, and that the wedge pressurecatheter was well placed.

[0140] After 50 minutes of blood-substitution below 10° C., the minimumbody temperature recorded was 2.9° C. Rewarming was then begun, andafter 28 minutes of warming, the animal's body temperature reached 10°C., and 750 ml of whole blood were added to the circuit, replacingsolution A.

[0141] Heartbeat was detected 8 minutes after blood was re-infused intothe animal. Over the next 30 minutes while the animal warmed, 10 ml ofNaHCO₃, were introduced i.v. and CaCl₂ (50 mg) was also injected i.v.,as was 80 mg of methyl prednisolone. Within a few minutes of adding theCaCl₂, massive clot formation was evident. It was thought that theblood, which was anti-coagulated with citrate, clotted as a result ofadding CaCl₂. The experiment was then discontinued.

[0142] In this experiment, the rate of flow of blood substitute throughthe animal and bypass circuit appeared high, while the left atrialpressure remained acceptably low. The factors which were thought tocontribute to this result were the use of nitroprusside, and themaintenance of a light anesthetic state during the cooling process. 1-2ml of heparin will be added to the blood prior to its re-introductioninto the animal. It is believed that heparinizing the re-introducedblood will eliminate the massive clotting which caused an unexpected endto this experiment.

Example 10

[0143] Ice-cold Blood Substitution of a Dog with Solution HLB.

[0144] Place a 25-30 Kg dog on partial cardio-pulmonary bypass. Surfaceand core cool the dog to near the ice point (1-3° C.). Replace the dog'sblood with solution HLB hypothermic blood substitute, described inExample 1. Retain the blood for transfusion during rewarming. Reduce theanimal's body temperature to near the ice point (below 4° C.) and thenrewarm. Replace the blood substitute with blood with warming and revivethe animal.

[0145] Preparation.

[0146] Catheterize the dog by means of the right radial vein, injectediv with pentothal, then fit with an endotracheal tube and ventilate withisofluorane (or Flether) in 100% O₂. Initiate a Ringer's lactate drip ata rate titrated to the dog's arterial blood pressure (approx. 40 ml/hriv). Place the dog on a cooling blanket cooled with recirculating icewater. Catheterize the right carotid artery to allow for blood pressure(CAP) monitoring, and add a 3-way stopcock in-line to allow arterialblood sampling every 10-60 min. throughout the entire procedure. Inserta foley catheter for urine collection and measure the urine volumethroughout the procedure. Implant a 2 lumen, 7 F, Swan Ganz wedgecatheter via the right jugular vein or right femoral vein, which is fedthrough the right heart into the pulmonary artery. Use the distal portto measure pulmonary wedge pressure (PAW), the proximal port is used forcentral venous pressure (CVP). (If necessary CVP may be measured with acatheter inserted in one of the brachial veins.) Isolate the leftfemoral artery and vein and prepare for cannulation. Heparinize theanimal (approx. 5,000 u). Insert a Biomedicus venous return cannula(15-19 F) in the femoral vein and a Biomedicus arterial cannula (12-15F) in the femoral artery. Measure the activated clotting time (ACT)every 45 min. (until blood substitution) and adjust the heparin suchthat it remains greater than 400 sec. Attach a thermocouple approx.midway to an esophageal tube and insert the unit so that the tube entersthe stomach. A second thermocouple is placed rectally. Attach ECG leads.Add Solu-Delta-Cortef (Upjohn, veterinary prednisolone Na succinate), 80mg by iv injection. Coat the eyes with Terrimycin (or Lacrylube), andadd DiGel (or Maalox, 20 ml) through the esophageal tube.

[0147] Measurements.

[0148] Measure arterial blood gasses, pH and hematocrit in every bloodsample, and in some cases electrolytes, enzymes and other chemistries.Monitor esophageal and rectal temperature as well as the arterial inflowand venous return blood temperatures. Monitor CAP, PAW, CVP, ECG, andairway pressure. Temperatures should be displayed digitally and storedas a function of time in a computerized data acquisition system. Thepressures and ECG should be displayed as real time waveforms or asnumerical data and stored by the computer.

[0149] Bypass Circuit Components.

[0150] The circuit features a Biomedicus centrifugal blood pump and flowmeter, a Terumo hollow fiber membrane oxygenator with built-in heatexchanger, Shiley hard shell venous reservoir with filter and asecondary heat exchanger with integral bubble trap (Electromedics)located as close to the animal as possible. A drain segment is locatednear the inlet of the venous reservoir and terminates with a checkvalve. This allows rapid and efficient blood/blood-substitute exchanges.There is an A-V shunt segment that allows circulation when not onbypass.

[0151] The venous reservoir can be filled from either the 1 literseparatory funnel through the “quick prime” port or from dual infusionbags through one of the cardiotomy ports. The arterial line from theoxygenator to the arterial cannula and the A-V shunt are constructedfrom {fraction (1/4)}″ tubing; the venous return, drain and pump-headlines are {fraction (3/8)}″. In those segments where severe bending canoccur, heavy-wall tubing is used or the tube is braced with “spiralwrap.”

[0152] The patient loop is double wrapped and the entire circuit (sansthe factory sterilized reservoir, secondary heat exchanger andoxygenator) is ethylene oxide gas sterilized as six basic sections(pump-head, flow meter section, central bypass loop, funnel, infusionline, and gas filter line).

[0153] Bypass Circuit Support.

[0154] Ice water, pumped from one of two 10 gal. insulated reservoirs,is used to cool the oxygenator and secondary heat exchangers. The otherreservoir supplies the cooling blanket. At the onset of surgery, icewater is circulated through the cooling blanket. At the onset of bypass,room temp. water is circulated through the circuit heat exchangers.

[0155] Temperature is slowly decreased by adding ice to the reservoir,in quantities sufficient to maintain a 7-10° C. difference between theesophageal and blood stream temperatures. After blood substitution (i.e.to a hematocrit of less than about 4%) full ice water flow is commenced.

[0156] Upon rewarming, ice is removed from the reservoir and the heateris activated. The temperature of the warming stream is limited to amaximum of 10° C. greater than the venous return temperature, by manualadjustment of the heater thermostat.

[0157] The oxygenator is supplied with sterile, filtered 100% O₂.

[0158] Blood Substitution.

[0159] The circuit is primed with 2 liters of solution L (Example 1),and recirculated through the A-V shunt to ensure temperature-gasequilibrium. The cannulas are attached to the arterial and venous linesof the bypass circuit, and the lines remain clamped. The cooling blanketis wrapped around the patient who is surface cooled until a deepesophageal temperature of 35° C. is reached.

[0160] The clamps are removed, and bypass is commenced with the solutionL-diluted blood stream at room temperature (approx. 25° C.). At theonset of cooling, gaseous anesthesia is discontinued, and the dog ismanaged with 2.5% pentothal.

[0161] The blood stream is gradually cooled until the animal has anesophageal temperature of 20° C., at which time blood is removed byclamping the venous return at the reservoir inlet and draining from thedrain segment while L solution is infused. During this exchange, anadditional 2 liters of L solution is added to the venous reservoir andwhen the level of L solution drops to 250 ml, approximately 6 liters ofHLB is added stepwise until all of the blood is removed (HCT less than2%, visual observation). Approximately 4 liters ofblood/blood-substitute mixtures collected in sterile bottles andretained for reinfusion. The very dilute blood mixture (about 5½ liters)is discarded.

[0162] After 4 liters have been exchanged (i.e. after the addition of 2liters of solution L and 2 liters of solution HLB), 20 meq KCl will beinjected via a stopcock on the secondary heat exchanger, to arrest theheart. During the exchange, the inflow is adjusted such that the PAW iskept below 5 mm Hg and the rate of efflux equals the rate of influx,i.e. as close to isovolemia as possible. At the end of the exchange thefinal reservoir level will be about 500 ml, the PAW below 5 mm Hg andthe CVP less than 5 mm Hg. Flow will be adjusted such that isovolemiawill be maintained (constant reservoir level and the above pressurelevels, i.e. PAW<5 mm Hg and CVP<5 mm Hg).

[0163] When almost all of the blood is removed (HCT less than 4%, visualobservation), the cooling stream can be reduced to ice water temperature(filling the reservoir with ice), and the dog rapidly cooled to itsminimum temperature. If the HCT is observed to rise at any time duringcold perfusion, the blood mixture can be removed by exchanging with 2 to4 liters of solution HLB by the method described above.

[0164] During the entire procedure, arterial blood samples are taken andblood gasses, pH, HCT, and in some cases electrolytes, and other bloodchemistries monitored.

[0165] After about 1-2 hours of blood substituted cooling, the dog'stemperature will be about 1-4° C., and rewarming will begin. The dogwill be rewarmed, by removing the ice from the supply reservoir andwarming its contents with the heater which in turn warms the blankets.When the esophageal temp reaches 15° C., 4 liters of solution L with 25g mannitol will be exchanged with the solution HLB followed by the 4liters of collected blood mixture. The effluent will be discarded.

[0166] The animal will be warmed gently, blood stream temperaturedifferential less than 10° C. and never above 40° C. The heart willspontaneously begin to beat. When the animal's temperatures (esophagealand rectal) reach about 35° C., physiological parameters are stabilized,and it can support itself, it can be weaned from the extracorporealcircuit.

Example 11

[0167] Reviving An Ice-cold Blood-substituted Dog.

[0168] A 26.8 kg male dog was anesthetized with nembutal and intubated.It was moved to the operating room, ventilated, and catheterized withvenous, Foley, arterial, and Swan-Ganz catheters, and after i.v.heparin, its right femoral artery and vein were cannulated. Anesophageal tube was inserted and antacid administered. Temperaturesensors were placed in the esophagus and the rectum. Methyl prednisolonewas injected i.v.

[0169] The animal was wrapped in a cooling blanket, and surface coolinginitiated. The animal's cannulas were connected to a bypass circuit,which consisted of a vortex blood pump, an oxygenator with a built-inheat exchanger, a secondary in-line heat exchanger, and a funnel for therapid administration of blood and blood substitute. Whole blood (225 ml)was removed from the dog and saved for rewarming. Blood volume wasquickly replaced with HLB solution. The bypass circuit containing 1.05liters of HLB solution was opened to the animal, and core cooling began.

[0170] Thirty three liters of blood substitute were exchanged. By thetime the ice-point was approached, the hematocrit was far below 1%. Theanimal's deep esophageal temperature was below 10° C. for 4 hours and 5minutes, with a minimum recorded temperature of 0.7° C. (Table 2).

[0171] Following the hypothermic period, the animal was warmed. Whenbody temperature climbed past 10° C., venous effluent and whole bloodpreviously collected, as well as donor blood, was returned to thecircuit; hematocrit increased with increasing temperature. Lidocaine andbicarbonate were administered, the heart defribillated, an ventilationbegun. When blood pressure and body temperatures approached normal, theanimal was weaned from bypass, and protamine and Lasix injected. Severalhours after warm-up, the animal was conscious and responsive. The animalremained alive and well after the procedure.

Example 12

[0172] Reviving an Ice-cold Blood-substituted Baboon.

[0173] A 24 kg male baboon of the species Papio annubis was anesthetizedfirst with ketamine and acepromazine i.m., then with i.v. pentothal. Itwas then immobilized with pancuronium bromide. It was intubated,ventilated, and catheterized with venous, Foley, and arterial catheters.The animal was wrapped in a cooling blanket, and surface coolinginitiated. After i.v. heparin was administered, the baboon's rightfemoral artery and bilateral femoral veins were cannulated. Temperaturesensors were placed in the esophagus, rectum and brain. The animal wasinstrumented for EKG, somatosensory evoked potentials (SSEPs) and EEG.Dexamethazone was injected i.v.

[0174] The animal's cannulas were connected to a bypass circuit, whichconsisted of a vortex blood pump, an oxygenator with a built-in heatexchanger, and a funnel for the rapid administration of blood and bloodsubstitute. Whole blood (300 ml) was removed from the baboon and savedfor rewarming. The volume was quickly replaced with 300 ml ofphysiological saline solution. The bypass circuit, containing 2 litersof Plasmalyte (commercially available electrolyte solution), was openedto the animal and core cooling begun.

[0175] After the deep esophageal temperature declined below 13° C.,another 2 liters of Plasmalyte containing 12.5 g of mannitol, was addedto the circuit, replacing the mixture of blood and Plasmalyte whichpreviously filled the circuit. This diluted blood was saved for useduring warming. Immediately afterwards, 10 liters of HLB solution wereadded, replacing the Plasmalyte. By the time the ice-point was reached,the hematocrit was far below 1%. When the animal reached braintemperature of 3.4° C. and deep esophageal temperature of 2.8° C., theblood pump was stopped and the animal was maintained under a conditionof circulatory arrest (standstill) for 45 minutes. After this period,circulation was resumed.

[0176] Following the hypothermic period, 4.2 liters of HLB solution wereadded to the bypass circuit, and the animal warmed. When bodytemperature reached 15° C., 2 liters of Plasmalyte were added to thecircuit to replace the HLB solution. Mannitol (6.25 g/l) was added tothe Plasmalyte in the circuit. Additionally, venous effluent and wholeblood previously collected, as well as donor blood cells andfresh-frozen plasma, were returned to the circuit; the animal'shematocrit increased with increasing body temperature. Another 12.5 g ofmannitol were added to the circuit. When the esophageal and rectaltemperatures approached normal, the heart fibrillated during warming andbegan beating. Ventilation was begun. When blood pressure and bodytemperatures approached normal, the animal was injected with protaminei.v., weaned from bypass, its cannulas and catheters removed, and itsincisions closed.

[0177] The animal's deep esophageal temperature had been below 15° C.for 3 hours, and below 10° C. for 2 hours 17 minutes, with a minimumrecorded temperature of 2.8° C. (Table 3). The following morning, theanimal was able to sit erect in its cage and pick up and eat pieces ofbanana, as well as drink apple juice. It remained alive and well untilsacrificed more than one week later for histological evaluation. TABLE 2REVIVAL OF AN ICE-COLD BLOOD-SUBSTITUTED DOG. MAP HR PAW CVP Flow TIMESOLUTION TE ° C. TR ° C. mmHg bpm mmHg mmHg L/min pH PCO₂ PO₂ Na K Hct11:57 am  36.1 12:21 pm  225 ml 35.2 129  133 12  3 HLB in & 225 mlblood out @ 12:19 pm 12:39 pm  32.6 34.8 134  141 12  3 12:40 pm  7.5225.1 403 144 2.7 34 12:52 pm  7.41 34.7 581 151 3.1 37 1:35 pm @ 1:3632.2 32.9 141  132 12  5 1.7 pm on bypass w/1.05 L HLB 1:40 pm 29.9 31.5115  128 10  3 1.7 1:43 pm 26.7 29.7 105  122 8 3 1.8 1:46 pm 7.36 37.1719 143 2.6 24 1:50 pm  5 L HLB 21.9 24.8 66  77 7 2 0.9  0 1:58 pm  4 LHLB 18.5 20.1 19 1.1 2:00 pm  4 L HLB 14.9 18.8 28 1.0 7.48 9.2 812 1552.5  0 2:02 pm 7.50 8.8 999+ 165 3.6  0 2:04 pm 10.4 16.9 37 1.5 2:05 pm9.9 16.2 37 2:08 pm  4 L HLB 8.6 15.3 37 1.5 2:14 pm 7.50 11.6 999+ 1594.2 2:16 pm  2 L HLB 5.7 12.3 27 1.5 2:20 pm 7.50 13.7 999+ 151 5.1 2:22pm 3.7 10.4 36 1.4 2:25 pm 3.3 9.8 35 1.6 2:27 pm 2.9 9.1 36 1 1.4 2:33pm 2.1 7.4 37 1.4 2:44 pm  2 L HLB 7.54 11.6 999+ 150 4.6 2:47 pm 1.44.8 36 3 1 1.3 2:50 pm 1.2 4.3 37 3 1 1.3 2:52 pm 1.2 4.2 37 3 1 1.32:59 pm  2 L HLB 1.1 3.4 21 0.6 3:55 pm 0.9 2.3 22 0.4 4:00 pm 7.63 9.6999+ 150 5.4 4:22 pm  3 L HLB 1.1 2.1 20 0.3 5:00 pm  2 L HLB 0.8 1.6 180.4 5:30 pm  3 L HLB 0.8 5:50 pm 7.48 11.0 999+ 150 5.7 5:56 pm 1.8 1.819 0.4 6:04 pm 4.7 2.8 27 1.0 6:06 pm  2 L HLB 6.6 3.3 27 1.1  0 6:08 pm 2 L Half 9.7 4.1 30 1.1 20 blood 6:09 pm 9.9 4.2 6:11 pm 10.7 5.3 31 18 7 1.0 6:12 pm 7.30 28.0 902 151 4.6 26 6:15 pm 13.8 6.7 30  24 13  21.1 6:25 pm 20.2 10.7 38 6 1 1.4 7.28 27.2 716 154 5.0 27 6:34 pm  1 Lblood 6:39 pm 7.34 38.9 670 158 3.2 26 6:42 pm 29.2 18.1 60 143 15  21.7 6:48 pm 7.37 28.9 587 154 2.9 27 6:57 pm 32.8 32.2 132  161 8 0 1.67:00 pm 7.33 27.3 496 150 2.7

[0178] TABLE 3 REVIVAL OF ICE--COLD BLOOD--SUBSTITUTED BABOON. MAP HRICP Flow Plas- TIME TE ° C. TR ° C. TB ° C. mmHg bpm mmHg L/min pH PCO₂PO₂ HLB malyte* Blood Hct 1:23 pm  1.6 L + (on ˜18   12.5 g bypass)mannitol 1:27 pm 31.3 32.8 33.2 60 83 9 2.2 1:30 pm 28.5 31.1 32.5 60 679 2.1 1:32 pm 23.4 29.0 30.9 50 45 8 2.2 1:35 pm 19.3 26.6 28.0 50 27 92.1 1:37 pm 18.0 25.5 26.5 50 24 11  2.2 1:38 pm 17.6 24.7 25.6 50 23 92.0 1:40 pm 16.8 23.7 24.3 50 25 8 2.0 1:44 pm 18.1 22.8 23.1 50 22 10 2.0 1:46 pm 18.0 22.2 22.3 50 8 2.1  0.3 L 1:50 pm  0.1 L 1:55 pm 12.219.3 18.2 50 4 7 L  0 2:02 pm 11.7 18.0 16.1 50 15  1.2 7.40 27 530 2 L2:05 pm 12.7 17.5 15.1 40 9 1.0 3 L 2:10 pm 11.3 16.9 14.1 40 9 1.3 2:14pm 10.5 16.2 13.3 50 11  1.3 7.34 17.1 578.6 2:21 pm 9.6 15.0 11.9 5011  1.3 2:25 pm 8.8 14.3 11.0 50 10  1.3 2:30 pm 7.9 13.4 9.9 50 10  1.37.37 21.2 782 2:40 pm 6.4 11.7 8.0 50 9 1.3 2:49 pm 5.3 10.4 6.7 55 10 1.2 2:54 pm 5.4 9.8 6.3 50 7 1.2 3:18 pm 3.9 8.6 4.6 50 7 1.0 3:29 pm3.2 7.8 3.8 50 8 1.0 3:32 pm 3.0 7.6 3.6 55 8 1.0 3:35 pm 2.9 7.4 3.5 507 1.0 3:37 pm 2.8 7.3 3.4 3 4:22 pm 3.7 10.1 4.8 1 4:24 pm 4.3 10.2 4.945 6 1.7 4:27 pm 6.5 10.4 6.4 55 8 1.0 2.2 L   4:32 pm 8.3 10.5 7.7 60 81.1 3 L 4:34 pm 9.0 10.6 8.5 65 10  1.0 4:36 pm 9.4 10.8 9.0 65 7 1.04:38 pm 9.9 10.9 9.4 60 6 1.0 4:39 pm 10.0 10.9 9.6 60 7 1.0 4:45 pm11.4 11.4 11.2 75 10  1.0 4:47 pm 11.9 11.6 11.9 80 9 1.0 4:51 pm 13.212.2 13.5 85 7 0.9 4:53 pm 14.1 12.6 14.6 85 slow 7 0.8 7.37 14 762 4:55pm 14.6 15.2 15.9 90 slow 6   2 L  0 4:59 pm    0.3 L 1/10 blood 5:01 pm    2 L 1/4  blood/    0.3 L 5:05 pm 18.0 15.3 18.1 55 7.33 22 224  25:16 pm +12.5 g manni- tol 5:20 pm 24.6 20.0 24.5 44 fib 12  2.1 5:24 pm   0.3 L plasma 5:25 pm 25.0 20.9 25.2 44 fib 13  2.0 7.30 25.3 593 5:36pm    0.4 L 12 blood +12.5 g manni- tol 5:37 pm 26.7 22.4 28.7 45 fib12  2.0 5:43 pm    0.3 L blood 5:55 pm 32.0 24.8 32.8 45 fib 10  2.25:57 pm 32.2 25.3 32.9 45 fib 8 2.2 6:10 pm 35.3 28.8 36.6 55 beat 11 6:13 pm 36.3 30.3 36.8  7 6:23 pm 37.3 33.7 36.2 60  7 7 1.3 7.34 28.2435 17 6:34 pm 7.39 31.9 322 20 6:36 pm    0.3 L plasma

[0179] The invention described above and claimed herein below embodiesnovel solutions that may be useful in a number of procedures. Thoseordinarily skilled in the art may be capable in light of the teaching ofthe specification and claims to make certain additions or modificationsto the invention without departing from the essence of the inventiondisclosed.

1. An aqueous-based blood substitute solution, wherein said solutionincludes an oncotic agent, and does not include more than 5 mM K⁺, anddoes not include a conventional biological buffer.
 2. The solution ofclaim 1 further comprising Na⁺ and an organic carboxylic acid, salt, orester thereof.
 3. A solution suitable for use as a blood substitute,comprising: 0-5 mM K⁺; concentrations of Na⁺, Mg⁺⁺, Ca⁺⁺, Cl⁻, which arephysiological or subphysiological concentrations; a macromolecularoncotic agent; an organic carboxylic acid, salt, or ester thereof; and asugar, with the proviso that said solution does not include more than 5mM K⁺, and with the further proviso that said solution does not includea conventional biological buffer.
 4. The solution of claim 3 wherein K⁺is present in the concentration range of 2-3 mM.
 5. The solution ofclaim 3 wherein Na⁺ is present in the concentration range of 130-150 mM.6. The solution of claim 3 wherein Mg⁺⁺ is present in the concentrationrange of 0.20-0.45 mM.
 7. The solution of claim 3 wherein Ca⁺⁺ ispresent in the concentration of 2.0-2.5 mM.
 8. The solution of claim 3wherein said sugar is a simple hexose sugar selected from the groupconsisting of glucose, fructose, or galactose, or a mixture thereof. 9.The solution of claim 3 wherein said organic carboxylic acid, salt orester thereof is represented by the formula RCOOX, wherein R is analkyl, alkenyl, or aryl, having a branched or straight chain containing1 to 30 carbons which carbons may be substituted; and X is a hydrogen orsodium or other biologically compatible ion substituent which can attachat the oxygen position, or is a short straight or branched chain alkylcontaining 1 to 4 carbons.
 10. The solution of claim 9 wherein saidorganic carboxylic acid is selected from the group consisting oflactate, acetate, pyruvate, or citrate.
 11. The solution of claim 3further comprising NaHCO₃.
 12. A method for maintaining a partially orsubstantially completely exsanguinated subject alive under hypothermicconditions, comprising substituting a solution comprising amacromolecular oncotic agent, Ca⁺⁺, and which does not contain aconventional biological buffer.
 13. The method of claim 12 furthercomprising glucose.
 14. A method for maintaining a partially orsubstantially completely exsanguinated subject alive under hypothermicconditions, comprising substituting a solution comprised of 0-5 mM K⁺,concentrations of Na⁺, Mg⁺⁺, Ca⁺⁺, Cl⁻ at physiological orsubphysiological levels, a macromolecular oncotic agent, an organiccarboxylic acid, salt, or ester thereof, a sugar, and NaHCO₃, with theproviso that said solution does not include more than 5 mM K⁺, and withthe further proviso that said solution does not include a conventionalbiological buffer.
 15. The method of claim 12 wherein said solution doesnot contain K⁺.
 16. A method for maintaining the biological integrity ofa subject or cells, tissues or organs from said subject, comprisingperfusing said subject or cells, tissues or organs from said subjectwith a solution comprising 0-5 mM K⁺, concentrations of Na⁺, Mg⁺⁺, Ca⁺⁺,Cl⁻ at physiological or subphysiological levels, a macromolecularoncotic agent, an organic carboxylic acid, salt or ester thereof, and asugar, with the proviso that said solution does not include more than 5mM K⁺, and with the further proviso that said solution does not includea conventional biological buffer.
 17. The method of claim 14 whereinsaid solution has substantially no ability to buffer pH in the range of7-8 in a cell-free system.
 18. The method of claim 14 wherein saidsolution does not contain a buffer selected from the group consisting ofHEPES, MOPS, TES, EPPS, THAM, or TRIS.
 19. A method for providing a heatsterilized blood substitute comprising: placing a solution comprised of0-5 mM K⁺, concentrations of Na⁺, Mg⁺⁺, Ca⁺⁺, Cl⁻ at physiological orsubphysiological levels, a macromolecular oncotic agent, an organiccarboxylic acid, salt or ester thereof, and a sugar, in aheat-sterilizable container; and raising the temperature of saidsolution under pressure and for a period of time sufficient to kill allor substantially all bacteria and inactivate all or substantially allviruses in the solution.
 20. The method of claim 17 wherein saidtemperature of said solution is raised under pressure to 120° C. for 15minutes.
 21. The method of claim 17 wherein said solution is placed inan autoclave.
 22. The method of claim 17 wherein said solution isinfused into a subject as a blood substitute or plasma extender.
 23. Themethod of claim 17 with the proviso that said solution does not includemore than 5 mM K⁺, and with the further proviso that said solution doesnot include a conventional biological buffer.
 24. A method for perfusinga subject prepared for circulatory perfusion in need thereof, comprisingthe steps of: reducing the subject's temperature to a temperature belownormal; circulating into the subject a solution comprising 0-5 mM K⁺,concentrations of Na⁺, Mg⁺⁺, Ca⁺⁺, Cl⁻ at physiological orsubphysiological levels, a macromolecular oncotic agent, an organiccarboxylic acid or salt thereof, a sugar, and NaHCO₃; and subsequentlyreturning blood to the subject.
 25. The method of claim 22 with theproviso that said solution does not include more than 5 mM K⁺, and withthe further proviso that said solution does not include a conventionalbiological buffer.
 26. The method of claim 22, comprising the additionalstep of placing the subject in a hyperbaric oxygen atmosphere afterreducing the subject's temperature to a temperature below normal, andfurther comprising the step of allowing the subject to regenerate anormal blood level in a hyperbaric oxygen atmosphere.